Hydroxymethylglutaryl-coenzyme A reductase (HMG-CoA reductase) is the key enzyme that controls the rate of cholesterol biosynthesis. HMG-CoA reductase is regulated by a variety of agents, the most notable of which is cholesterol itself. When dietary or exogenous cholesterol is administered to cells, HMG-CoA reductase activity declines and endogenous cholesterol biosynthesis ceases. This decline, as well as those caused by other agents, result primarily from changes in the concentration of HMG-CoA reductase protein. The long-term goal of this project is to decipher the molecular mechanism by which cholesterol regulates HMG-CoA reductase. Knowledge of this regulatory mechanism is important because aberrations result in over-production of cholesterol, which results in atherosclerosis and death. Evidence suggests that cholesterol regulates expression of the HMG-CoA reductase gene. To explore this possibility, a probe specific for reductase gene sequences is required. The initial goal is therefore to clone the gene for HMG-CoA reductase. Two independent cloning strategies have been devised -- one involves DNA-mediated gene transfer and plasmid rescue and the other involves isolation of mRNA from cells with amplified copies of the HMG-CoA reductase gene. Once isolated and characterized, the cloned gene will be used as a probe (1) to determine whether cholesterol regulates the level of HMG-CoA reductase mRNA, (2) to characterize the structure of the HMG-CoA reductase gene and (3) to attempt to identify putative regulatory sequences that might interact with cholesterol or protein-cholesterol complexes.